Heterologous expression of pyrroline-5-carboxylate reductase ana L-Lysine-6-dehydrogenase genes for L-pipecolic acid production

Abstract

L-pipecolic acid (L-PA) is a non-protein amino acid. It is an important precursor of many useful microbial secondary metabolites such as an immunosuppressant rapamycin. This research aims to produce L-PA by coupling reaction of lysine 6-dehydrogenase (Lys 6-DH) and pyrroline-5-carboxylate reductase (P5CR). The lys 6-dh gene from Acromobacter denitrificans was already cloned into Escherichia coli BL21(DE3) in previous research. Therefore, in this study proC encoding P5CR from Bacillus cereus ATCC 11778 was cloned into an expression vector pET-17b and then transformed to E. coli BL21(DE3). The optimum condition for proC expression was induction with 0.4 mM IPTG for 4 hours. The enzyme was purified 2.25 fold by DEAE-Toyopearl and Butyl-Toyopearl column chromatographies with 14.9% yield. The molecular mass of enzyme subunit was about 28.9 kDa. The apparent Km for L-proline and NAD+ were 1 and 1.54 mM, respectively. The obtained proC was co-expressed with the lys 6-dh in E. coli BL21(DE3) in order to produce L-PA. The production of L-PA, approximately 1.74 g/l, was obtained by induction of the recombinant clone with 0.1 mM IPTG for 4 hours and then 0.05 g of cell wet weight was incubated in the reaction mixture consisted of 200 mM L-lysine in 200 mM Tris-HCl buffer, pH 9.0 for 24 hours.